In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11β hydroxylase; P450c11). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line, H295R, was utilized in this study to examine the intracellular second messenger pathways regulating expression of P450aldo and P450c11, using specific ribonuclease protection assays. Treatment of H295R cells with angiotensin II or potassium (K⁺) caused a time-dependent induction in the level of P450aldo transcripts. While K⁺ treatment was more specific for the induction of P450aldo mRNA, treatment with angiotensin II increased levels of both P450aldo and P450c11 transcripts. To define the second messenger systems which influence transcript levels for these enzymes, the effects of agonists of the protein kinase A, protein kinase C, and calcium pathways were tested on the expression of P450aldo and P450c11. Activation of the protein kinase A pathway by the agonists, dibutyryl cAMP or forskolin, preferentially increased the P450c11 transcript to a greater degree than P450aldo. Interestingly, activation of the protein kinase C pathway by tetradecanoylphorbol acetate (TPA) did not alter transcripts for either P450aldo or P450c11. The calcium channel agonist BAYK 8644 mimicked the effects of K⁺ by increasing the transcript for P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K⁺ on the levels of P450aldo transcripts without affecting the stimulatory effect of dbcAMP. This study demonstrates that the protein kinase A pathway preferentially induces P450c11 mRNA over that of P450aldo. In addition, pharmacologic agents that affect calcium levels provide evidence for an additional regulatory mechanism in modulating the expression of P450aldo. This is of importance since the major physiologic regulators of aldosterone secretion, angiotensin II and K⁺ are able to increase intracellular calcium but have little effect on intracellular cAMP levels.